Release of Intracellular Stores Can Hyperactivate Catsper1 and Catsper2 Null Sperm ⁎ Becky Marquez, George Ignotz, Susan S

Developmental Biology 303 (2007) 214–221 www.elsevier.com/locate/ydbio

Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm ⁎ Becky Marquez, George Ignotz, Susan S. Suarez

Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Received for publication 13 May 2006; revised 2 November 2006; accepted 3 November 2006 Available online 10 November 2006

Abstract

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca2+, which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca2+ from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca2+ was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 μM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20–40% of sperm, induced hyperactivation. Addition of 1 mM Ni2+ diminished the response. This suggests that intracellular Ca2+ is abnormally low in the null sperm flagella. When intracellular Ca2+ was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca2+ is required to supplement store-released Ca2+ to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca2+ entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility. © 2006 Elsevier Inc. All rights reserved.

Keywords: Sperm; Sperm motility; Flagellum; Calcium; Calcium channel; Calcium store; CatSper

Introduction fertilization, the high-amplitude flagellar bends of hyperactivation are required by sperm to penetrate the oocyte zona Ca2+ signaling in sperm is critical for fertilization. Ca2+ pellucida (Stauss et al., 1995; Quill et al., 2003). uptake (Handrow et al., 1989) is associated with capacitation, a The mechanisms that trigger the rise of intracellular Ca2+ to process whereby mammalian sperm gain the capacity to initiate and maintain hyperactivation are not completely under- undergo the acrosome reaction and fertilize oocytes (Yanagi- stood, but increased Ca2+ entry via plasma membrane channels machi, 1994). Activation of motility occurs when sperm are is known to play a key role. Voltage-gated Ca2+ channels released from the cauda epididymidis. Activated sperm swim in (Trevino et al., 2004; Wennemuth et al., 2000), cyclic linear trajectories generated by moderate-amplitude symmetri- nucleotide-gated Ca2+ channels (Wiesner et al., 1998), and cal flagellar beating (Suarez et al., 1983). Elevation of flagellar canonical transient receptor potential (TRPC) channels (Trevino Ca2+ during capacitation induces hyperactivation (Suarez and et al., 2001; Castellano et al., 2002) have been localized to the Dai, 1995; Ho and Suarez, 2001) by increasing the amplitude of flagellum. However, only sperm-specific proteins CatSper1 the principal flagellar bend, which produces asymmetrical (Ren et al., 2001) and CatSper2 (Quill et al., 2003) have been beating (Ho et al., 2002). Hyperactivated sperm exhibit frequent shown to be required for male fertility. These CatSper proteins changes in direction that may enable them to navigate the form alkaline-sensitive voltage-gated Ca2+ channels that are tortuous lumen of the oviduct to reach the oocyte. During located on the principal piece of the flagellum (Ren et al., 2001; Quill et al., 2001; Kirichok et al., 2006). Sperm deficient in 2+ ⁎ Corresponding author. Fax: +1 607 253 3541. CatSper1 or CatSper2 lack depolarization-evoked Ca entry E-mail address: [email protected] (S.S. Suarez). (Carlson et al., 2003, 2005). Although it has not been

0012-1606/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2006.11.007 B. Marquez et al. / Developmental Biology 303 (2007) 214–221 215 demonstrated that CatSper1 and CatSper2 form a single Sperm lysates representing 106 sperm were reduced by heating for 3 min at – channel, disruption of either gene prevents the expression of 60°C in the presence of 40 mM DTT, resolved by SDS PAGE (Laemmli, 1970), and transferred to nitrocellulose (Towbin et al., 1979). Blots were blocked for the other protein in the flagellum (Carlson et al., 2005). Sperm 1 h in Tris-buffered saline containing 0.5% Tween-20 and 2.5% non-fat dry lacking CatSper show abnormally high flagellar beat frequency milk. CatSper2 polyclonal antibody, directed against the carboxy terminal and low bend amplitude, as well as a failure to hyperactivate epitope (Quill et al., 2001) and provided by Dr. Timothy Quill (UT during capacitation (Carlson et al., 2003, 2005; Quill et al., Southwestern) was applied at 1:3000 dilution overnight at 4°C. After rinsing, 2003). blots were incubated with HRP-conjugated goat anti-rabbit IgG and were developed using enhanced chemiluminescence (SuperSignal West Pico, Pierce, There is also evidence for a mechanism in which release of 2+ Rockford, IL). To assess sample loading, blots were stripped and re-probed Ca from an IP3-gated internal store at the base of the flagellum using anti-tubulin and a goat anti-mouse IgG–HRP conjugate. Anti-β-tubulin initiates hyperactivation (Ho and Suarez, 2001). IP3 receptors (mAbE7) developed by Klymkowsky was obtained from the Developmental have been localized to a portion of the redundant nuclear Studies Hybridoma Bank developed under the auspices of the NICHD and envelope on the side of the flagellum which increases bend maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242. amplitude in response to Ca2+ (Ho and Suarez, 2003). Because each flagellar bend originates at the base of the flagellum and is Sperm preparation and experimental conditions propagated down to the principal piece, it is therefore proposed 2+ that the store provides the initial Ca signal, which somehow Sperm were collected as previously described (Suarez and Osman, 1987). stimulates influx through CatSper channels in the principal Briefly, cauda epididymides were punctured with a 27-gauge needle and piece. incubated at 37°C to allow sperm to disperse into surrounding medium. After 10 min, sperm were aspirated and placed in a prewarmed 1.5 ml microcentrifuge The purpose of this study was to investigate the 6 2+ tube. Sperm numbers were adjusted to 5×10 /ml and sperm were treated with contributions of extracellular and intracellular Ca to the 50 μM thimerosal or 5 mM procaine immediately or were incubated for 90 min initiation and maintenance of hyperactivated motility. Using under capacitating conditions at 37°C with 5% CO2. CatSper1 and CatSper2 null sperm, we investigated whether 2+ release of Ca from an internal store can initiate hyperactiva- Analysis of sperm motility tion and if an extracellular Ca2+ source is required to sustain hyperactivation. Aliquots of sperm were placed in slide chambers of 50 μm depth and motility was examined on a 37°C stage of a Zeiss Axiovert 35 microscope using differential interference contrast optics with a 40× objective (Carl Zeiss, Inc., Materials and methods Thornbrook, NY) and stroboscopic illumination at 60 Hz provided by a 75 W xenon flash tube (Chadwick-Helmuth Co., El Monte, CA). Videotaping was Chemicals and medium conducted using a black-and-white Dage CCD 72 video camera (Dage-MTI, Inc., Michigan City, IN) connected to a Panasonic AG-7300 Super VHS All chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO) with videocassette recorder (Panasonic Industrial Co., Secaucus, NY). Videotapes the following exceptions. HEPES, BSA, and 1,2-bis(2-aminophenoxy)ethane- were used to assess the swimming patterns of sperm. For each treatment sample, N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) were obtained 100–110 motile sperm were analyzed and, for each experiment, replicate tests from Calbiochem Corporation (La Jolla, CA). Molecular Probes (Eugene, OR) were performed using three males from each genotype. provided fluo3 AM and BAPTA. Sperm motility was also evaluated using computer-assisted sperm analysis The medium consisted of 110 mM NaCl, 2.68 mM KCl, 0.36 mM NaH2PO4, (CASA). Sperm movement was imaged using a 4× Olympus negative phase 25 mM NaHCO3, 25 mM HEPES, 2.4 mM CaCl2, 0.49 mM MgCl2, 5.56 mM objective (Hi-Tech Instruments, Philadelphia, PA) and recorded using a glucose, 1 mM pyruvate, 0.006% Na penicillin G, and 20 mg/ml BSA (pH 7.4, Panasonic AG-7300 Super VHS video recorder. The video images were 290–300 mosM/kg) (Suarez and Osman, 1987). digitized (30 frames at 60 Hz) and analyzed using HTM-IVOS (Version 10, Hamilton Thorne Biosciences, Beverly, MA). Motion parameters measured Animals were curvilinear velocity (VCL, the rate of travel of the sperm head), amplitude of lateral head displacement (ALH, degree of side-to-side head movement CatSper1 and CatSper2 null mice were provided by Drs. Timothy Quill and measured as the mean width of head oscillations), and beat/cross frequency David Garbers (UT Southwestern) and were derived as previously described (BCF, number of times the head crosses the averaged path per second). Because (Carlson et al., 2003; Ren et al., 2001; Quill et al., 2001, 2003). Males movement of the sperm head depends on flagellar activity, it can be used as an heterozygous (+/−) for CATSPER1 or CATSPER2 were used as controls in indicator of flagellar bending patterns. Increased VCL and ALH are indicative of experiments involving males homozygous (−/−) for the disrupted genes, hyperactivation (Mortimer and Mortimer, 1990). For each treatment sample, respectively, and wild-type (C57BL/6) males were used when testing the effects 400–450 motile sperm were analyzed and for each experiment, replicate tests of BAPTA or BAPTA-AM on motility. were performed using three males from each genotype.

Immunoblotting

Cauda epididymides were recovered from three male mice each of genotypes CATSPER2 +/+, +/−, and −/−, placed in 1 ml warmed (37°C) PBS in a small culture dish, and nicked at several sites with small scissors to disperse sperm. After 15 min of incubation, tissue was removed and the sperm-containing supernatants were transferred to Eppendorf tubes. An aliquot of each was removed for hemacytometer counting, and sperm were pelleted by centrifuga- Fig. 1. Immunoblots demonstrating that sperm from CATSPER2+/− males tion. Sperm pellets were suspended in non-reducing SDS sample buffer at a contain the same levels of CatSper2 protein as sperm from wild-type concentration of 10×107 sperm/ml and heated at 60°C for 5 min. Lysates were (CATSPER2+/+) males. Upper blot, sperm extracts probed with CatSper2 centrifuged to remove insoluble material and supernatants were stored at −80°C antibody. Lower blot, after removing CatSper2 antibody, blots were probed with until used for SDS–PAGE. anti-tubulin antibody to indicate numbers of sperm loaded per lane. 216 B. Marquez et al. / Developmental Biology 303 (2007) 214–221

Table 1 confirmed that sperm from CATSPER2−/−males lacked Cat- 2+ a CASA measurements of effects of Ca and thimerosal on sperm motility Sper2 protein (Carlson et al., 2005) and that sperm from − Motile (%) VCL (μm/s) ALH (μm) BCF (/s) CATSPER2+/ males contained the same amount of CatSper2 +/+ Control 83.0±2.1 331.8±10.2 14.8±0.6 27.5±0.5 protein as sperm from CATSPER2 males (Fig. 1). +BAPTA 83.3±1.9 265.6±3.6 b 11.6±0.4 b 31.8±0.4 b Thimerosal 75.7±6.3 392.0±15.0 c 23.2±0.2 c 23.6±2.5 2+ c c c Release of Ca from internal stores is sufficient to initiate +BAPTA 78.0±1.2 391.9±4.1 24.4±0.5 20.8±1.4 hyperactivation in wild-type sperm a Sperm were incubated in medium containing 2.4 mM Ca or 1.2 mM Ca+ μ 10 mM BAPTA in the absence or presence of 50 M thimerosal for 1 min Thimerosal (50 μM) induced 90–100% of motile wild-type (mean ce:hsp sp="0.12"/>±SEM). b Different from all other treatments (P<0.05). sperm to hyperactivate (Supplemental Movie 1B) compared to a c Different from control (P<0.05). 5–10% baseline in controls (Suppental Movie 1A). This effect was detected by CASA as increases in mean ALH, which indicates increased flagellar bend amplitude, and VCL (Table Ca2+ image analysis 1). After 5 min exposure to thimerosal, motility declined and sperm arrested with curved flagellum (Fig. 2). Stimulation by 6 μ 2+ Sperm (5×10 /ml) were loaded with 5 M of the fluorescent Ca indicator thimerosal was not blocked by lowering extracellular Ca2+ with dye fluo3 AM (in 0.5% DMSO) for 40 min at 37°C in medium lacking bicarbonate and containing 0.6% BSA. Extracellular dye was removed by 10 mM BAPTA to approximately 30 nM (calculated using centrifugation at 170×g for 5 min. Sperm were resuspended in medium and MaxChelator: www.stanford.edu/~cpatton/maxc.html), which 2+ incubated for an additional 20 min to allow de-esterification of the dye into its is below the 50 nM intracellular Ca level of non-hyperacti- charged, Ca2+-sensitive form. The fluorescence of individual motile sperm in an vated sperm with normal activated (i.e., progressive) motility open micro-chamber at 37°C (PDMI-2 Micro-Incubator, Medical Systems Corp. (Suarez and Dai, 1995; Ho et al., 2002; Table 1). Greenvale, NY) was monitored before and after application of treatments. Fluorescent imaging of fluo3-loaded sperm demonstrated Sperm were tethered by the head to a glass coverslip in the floor of the chamber 2+ by adding them to the chamber in medium lacking BSA, then flooding the that thimerosal raised Ca in the head and flagellum in less chamber with complete medium. Fluorescence intensity was detected with an than 25 s (Fig. 3). epifluorescence microscope using a 480±40 nm excitation filter, 535±50 nm emission filter, and a 505 nm long-pass dichroic mirror (Chroma Technology Elevation of sperm Ca2+ overcomes the motility defect in Corp., Rockingham, VT) with an oil immersion 40× Fluar objective (n.a. 1.3, Carl Zeiss Inc.). Stroboscopic illumination was provided by a 75 W xenon arc CatSper1 or CatSper2 null sperm flash lamp. Images were captured with a Sensicam High Performance camera (The Cooke Corporation, Auburn Hills, MI) and digitized at 2 images/s Analysis of video recorded images of free-swimming sperm controlled by IPLab Spectrum software (Signal Analytics, Vienna, VA). revealed abnormal activated motility in both CatSper1 and CatSper2 null sperm. CATSPER1−/− sperm swam in circular Data analysis trajectories instead of normal linear trajectories. The flagella generated curves of long wavelength and moderate-amplitude Data were analyzed using Minitab statistical software (Minitab Inc., State College, PA). Treatment effects were detected using analysis of variance, in the direction of the curvature of the head (i.e., the principal followed by Tukey's test for individual post-hoc comparisons and were bends) and minimal reverse bends (Fig. 4C and Supplemental − − considered statistically significant when P<0.05. Movie 2A, D). The flagellar beating patterns of CATSPER2 / sperm were similar to those of CATSPER1−/− sperm, but the Results sperm swam in straighter paths, primarily due to periodic rolling along the long axis (Fig. 4D, Supplemental Movie 3A, D). CatSper protein content in sperm from null and heterozygous These movement patterns were evident immediately after males extraction of sperm from epididymides and were maintained throughout a 90-min incubation under capacitating conditions. Western blot analysis of sperm samples from heterozygous Exposure of CatSper null sperm to 50 μM thimerosal visibly and homozygous null males with anti-CatSper2 antibody decreased the asymmetry of the flagellar beat while increasing

Fig. 2. Flagellar curvature generated by thimerosal. Activated sperm swimming in medium alone (A). Prolonged exposure to thimerosal caused sperm to arrest with the flagellum curved in the direction opposite to the curve of the head (B). Scale bar=10 μm. B. Marquez et al. / Developmental Biology 303 (2007) 214–221 217

Fig. 3. Ca2+ imaging of fluo3-loaded sperm. Images of wild-type sperm were captured before (A, C) and 25 s after (B, D) application of medium alone (B) or containing thimerosal (D). Warmer colors indicate higher fluorescent intensities and increased intracellular Ca2+. Scale bar=10 μm. the bend amplitude (Supplemental Movies 2B, E and 3B, E), activity to resemble that of normal, activated CATSPER+/− detected by CASA as increased ALH and VCL (Tables 2 and 3). sperm. In addition, 20–40% of the CATSPER1−/− and This produced straighter swimming trajectories in most sperm. CATSPER2−/− sperm hyperactivated in response to thimerosal, The beat frequencies of CATSPER1−/− (Table 2) and CATS- albeit to a lesser degree than CATSPER+/− sperm, of which 90– PER2−/− (Table 3) sperm were decreased, causing flagellar 100% hyperactivated. These responses of the null sperm were detected by CASA as increased mean ALH and VCL (Tables 2 and 3). Addition of 1 mM Ni2+ reduced the proportion of both CATSPER2+/− and CATSPER2−/− sperm exhibiting hyperactivated motility (Fig. 5). Procaine (5 mM) stimulated hyperactivation in 90% of CATSPER+/− sperm but did not hyperactivate CATSPER1−/− (Table 2 and Supplemental Movie 2C, F) or CATSPER2−/− (Table 3 and Supplemental Movie 3C, F) sperm.

Reduction of intracellular Ca2+ increases beat frequency and decreases flagellar bend amplitude

Incubation of wild-type sperm for 90 min under capacitating conditions led to the development of hyperactivation, detected by CASA as increased mean ALH and VCL (Table 4). When Ca was omitted from the medium, sperm had lower VCL and ALH as well as higher BCF, and only 17.0±1.2% of sperm hyperactivated compared to 63.3±3.2% in the presence of 2.4 mM added Ca. To further reduce intracellular Ca2+, wild-type sperm were exposed to a membrane permeable Ca2+ chelator, BAPTA-AM. This treatment further dampened the flagellar bend amplitude (ALH) and caused a greater increase in BCF (Table 5) while maintaining progressive sperm motility.

Discussion Fig. 4. Flagellar beating patterns of CATSPER1−/− and CATSPER2−/− sperm. +/− +/− CATSPER1 (A) and CATSPER2 (B) sperm display normal activated mo- All sperm from CATSPER+/− mice behaved as though they tility consisting of moderate-amplitude flagellar bends and nearly symmetrical beating. CATSPER1−/− (C) and CATSPER2−/− (D) sperm display low bending had inherited the wild-type CATSPER gene, because gene amplitude and slightly asymmetrical flagellar beating (D). Black and gray products are shared through cytoplasmic bridges in developing indicate the principal and the reverse flagellar bends, respectively. germ cells (Braun et al., 1989; Ventela et al., 2003). Nearly all 218 B. Marquez et al. / Developmental Biology 303 (2007) 214–221

Table 2 CASA measurements of CATSPER1 sperm treated with thimerosal or procaine a Motile (%) VCL (μm/s) ALH (μm) BCF (/s) +/−−/− +/−−/− +/−−/− +/−−/− Control 69±1 68±3 306.7±12.0 214.6±0.5 b 14.7±0.9 9.6±0.3 b 30.0±0.3 36.4±1.4 b Thimerosal 69±3 64±2 346.9±10.0 c 289.7±5.3 b, c 20.1±0.6 c 18.4±0.2 b, c 22.4±2.0 c 25.0±1.5 c Procaine 62±2 61±2 381.7±2.1 c, d 216.6±4.8b,d 18.6±0.5 c,d 9.0±0.1b,d 33.9±1.7 c,d 39.7±2.3b,d a Sperm were incubated in medium alone or containing 50 μM thimerosal or 5 mM procaine for 1 min (mean±SEM). b Different from +/− (P<0.05). c Different from control (P<0.05). d Different from thimerosal (P<0.05). sperm from the heterozygotes hyperactivated in response to internal stores and not an external Ca2+ entry pathway in procaine, rather than half, as would be expected if the CAT- providing Ca2+ to initiate hyperactivation. SPER gene products were not shared. The levels of CatSper2 Interestingly, procaine did not stimulate hyperactivation in protein, and presumably CatSper1 protein, were the same in CATSPER1−/− or CATSPER2−/− sperm, consistent with the sperm from heterozygotes as from wild-type males (Fig. 1). It dependence of procaine-induced hyperactivation on extracel- had previously been demonstrated that disruption of either lular Ca2+ (Marquez and Suarez, 2004). This suggests that CATSPER1 or CATSPER2 genes resulted in the absence of both procaine activates Ca2+ channels at the plasma membrane. CatSper1 and CatSper2 proteins in the sperm flagellum Previous analysis of bending in the midpiece indicated that (Carlson et al., 2005). procaine increases flagellar beat asymmetry of tethered The results presented here underscore the importance of CATSPER2−/− sperm held at room temperature, although not extracellular Ca2+ in the regulation of both activated and in a manner identical to hyperactivation (Carlson et al., 2005). hyperactivated motility. Release of Ca2+ from internal stores Our CASA data and observations of movement patterns of free- induced the transition from activated to hyperactivated motility swimming sperm at 37°C (mouse body temperature) did not in CATSPER+/− and CATSPER−/− sperm, but extracellular Ca2+ reveal hyperactivation in CATSPER2−/− sperm in response to was required to sustain hyperactivation. Moreover, lack of procaine. Nonetheless, the ability of thimerosal to induce hy- CatSper 1 and 2 resulted in flagellar activity that resembled the peractivation in the null sperm agrees with the assertion that flagellar movements of wild-type sperm treated to lower these sperm are capable of responding to intracellular Ca2+ intracellular Ca2+; however, motility resembling that of normal signals and modulating flagellar activity accordingly. activated wild-type sperm could be elicited in the null sperm It is evident that extracellular Ca2+ is required to maximize when internal Ca2+ was elevated by release of stores. and sustain hyperactivation because, without added Ca in the Release of internal store Ca2+ was sufficient to initiate medium, wild-type sperm showed only minimal development hyperactivation in wild-type sperm. Similar to the results from of hyperactivation over time. Additionally, 1 mM Ni2+ reduced bull sperm (Ho and Suarez, 2001), thimerosal quickly induced hyperactivation elicited by thimerosal in CATSPER2+/− and hyperactivation in the majority of wild-type mouse sperm, even CATSPER2−/− sperm. High Ni2+ doses have been shown to in the absence of available extracellular Ca2+. Thimerosal is block store-operated channels (SOC) in sperm (O'Toole et al., known to stimulate release of Ca2+ from internal stores 2000; Yoshida et al., 2003). Emptying of store Ca2+ is thought 2+ 2+ (Miyazaki et al., 1992). Ca stores gated by IP3R have been to trigger Ca influx to refill the store, presumably through identified at the acrosome (Walensky and Snyder, 1995; Herrick SOC at the plasma membrane (Putney, 1986; Berridge, 1995; et al., 2005) and base of the flagellum (Ho and Suarez, 2001; Ho Berridge et al., 2000). TRPC channels are candidates for SOC and Suarez, 2003), which correspond to the sites of increased (Clapham, 2003). It has already been demonstrated that TRPC2, Ca2+ seen in response to thimerosal in our experiments. which is localized to the sperm head, provides sustained Ca2+ Additionally, hyperactivation was elicited by thimerosal in entry during the zona pellucida-induced acrosome reaction mouse sperm lacking CatSper1 and 2, further implicating (Jungnickel et al., 2001). Furthermore, TRPC1 and TRPC3

Table 3 CASA measurements of CATSPER2 sperm treated with thimerosal or procaine a Motile (%) VCL (μm/s) ALH (μm) BCF (/s) +/−−/− +/−−/− +/−−/− +/−−/− Control 75±1 63±2 348.5±5.0 265.1±9.1 b 16.8±0.4 12.0±0.5 b 29.3±0.2 40.0±0.2 b Thimerosal 62±3 68±1 415.4±3.4 c 359.1±9.1 b, c 23.5±1.0 c 18.1±0.5 b, c 20.8±1.9 c 25.5±4.7 c Procaine 60±1 64±1 432.2±6.1 c, d 307.0±7.7 b, c, d 22.2±0.4 c 12.5±1.0 b, d 30.9±0.7 c, d 44.1±0.1 b, c, d a Sperm were incubated in medium alone or containing 50 μM thimerosal or 5 mM procaine for 1 min (mean±SEM). b Different from +/− (P<0.05). c Different from control (P<0.05). d Different from thimerosal (P<0.05). B. Marquez et al. / Developmental Biology 303 (2007) 214–221 219

Table 5 CASA measurements of effects of reduced intracellular Ca2+ on sperm motility a Motile (%) VCL (μm/s) ALH (μm) BCF (/s) Control 58.3±1.9 346.9±6.6 16.7±0.5 27.3±1.1 BAPTA-AM 65.3±3.5 198.7±4.0 b 8.7±0.4 b 37.7±0.6 b a Sperm were incubated in medium containing 0.25% DMSO alone or 25 μM BAPTA-AM in 0.25% DMSO for 15 min (mean±SEM). b Different from control (P<0.05).

rise in intracellular Ca2+ apparently overshot the amount needed to produce normal progressive, activated motility and caused them to hyperactivate. Intracellular Ca2+ is approximately 30–50 nM in sperm Fig. 5. Ni2+ reduced thimerosal-induced hyperactivation. CATSPER2+/− or − − exhibiting activated motility but 200–1000 nM in hyperacti- CATSPER2 / sperm were treated with thimerosal after 5 min pre-incubation in 1mMNi2+. Bars labeled with different letters indicate significant differences vated sperm (Suarez and Dai, 1995; Ho et al., 2002). Therefore, between treatments (P<0.05). the decreased effectiveness of thimerosal in sperm lacking CatSper1 or CatSper2 could be because cytosolic Ca2+ is maintained at lower levels than normal, making the extent of have been localized to the midpiece and principal piece, Ca2+ elevation required to initiate hyperactivation even greater. respectively; of mouse sperm (Trevino et al., 2001)and Whereas both CATSPER1−/− and CATSPER2−/− sperm therefore depletion of store Ca2+ could activate local SOC in were observed to express a similar mildly asymmetrical the flagellum to promote hyperactivation. There is also evidence flagellar beating, CATSPER1−/− sperm swam in circles on the indicating that chemotactic behavior, which consists of slides while CATSPER1−/− swam in general straight trajec- asymmetrical flagellar movements in ascidian sperm, is induced tories due to rolling on the longitudinal axis. The greater by a SOC-mediated increase in intracellular Ca2+ (Yoshida et tendency of CatSper1 null sperm to be captured by the al., 2003). surface of the glass slide is likely due to its more subdued Elevation of intracellular Ca2+ overcame the motility defect movement, exemplified as apparently lower VCL, ALH, and in CatSper1 and 2 null sperm. Sperm from CATSPER1−/− and BCF (Tables 2 and 3). Capture of sperm by flat surfaces CATSPER2−/− males expressed abnormal activated motility suppresses torsion in the neck and confines flagellar waves to consisting of low flagellar bend amplitude and higher beat a single plane; that is, it suppresses rolling (Woolley, 2003). frequency, as previously reported (Carlson et al., 2003, 2005). Because CATSPER1 null sperm lack CatSper2 protein and We show here for the first time that the abnormal activity might vice versa (Carlson et al., 2005), the differences in the be explained by abnormally low cytoplasmic Ca2+.The vigor of movement seen in sperm from CATSPER1 and abnormal motility could be corrected by thimerosal, presumably CATSPER2 nulls and heterozygotes are attributable to by releasing store Ca2+ to raise cytoplasmic levels. differences in genetic background. In the second report on Ca2+ acts through calmodulin on the axoneme to modulate the phenotype of CatSper1 null sperm (Carlson et al., 2003), flagellar curvature (Ho et al., 2002) and blocking the effects of the authors noted that the sperm were more vigorous than Ca2+ with calmodulin inhibitor W13 produces a similar pattern those described in first report (Ren et al., 2001) and they as seen in the CatSper null sperm (Aoki et al., 1999; Tash and attributed the difference to changes in genetic background due Means, 1982). Moreover, detergent-demembranated sperm to backcrossing with mice from another strain. display curved flagella with low amplitude bending and higher Ca2+ entry through a CatSper2-dependent pathway has been beat frequency when reactivated under Ca2+-deficient condi- proposed to influence basal cAMP levels because higher basal tions (Lindemann and Goltz, 1988; Lindemann et al., 1987). cAMP is associated with accelerated flagellar beat frequency of Hence, CATSPER1−/− or CATSPER2−/− sperm are likely to CATSPER2−/− sperm (Carlson et al., 2005). It is unclear how contain lower basal cytosolic Ca2+ in the flagellum such that cAMP generates such a flagellar response when experiments thimerosal raised cytosolic Ca2+ to the level required to support using detergent-permeabilized sperm indicate that flagellar normal activated motility. In some thimerosal-treated sperm, the curvature and beat frequency of motile sperm are not dependent on cAMP concentrations (Ho et al., 2002). Nevertheless, cAMP Table 4 signaling of phosphorylation is critical for initiation of motility CASA measurements of effects of extracellular Ca2+ on capacitation-induced (Somanath et al., 2004). It is interesting that, unlike sperm of hyperactivation a mutant mice lacking soluble adenylyl cyclase (Hess et al., 2005; Ca (mM) Motile (%) VCL (μm/s) ALH (μm) BCF (/s) Esposito et al., 2004), sperm from CatSper1 and CatSper2 null 2.4 59.7±0.9 366.8±8.1 18.5±0.3 21.7±1.0 males were highly motile when extracted from the epididymi- 2+ 0.0 71.3±1.2 b 256.3±4.5 b 13.8±0.5 b 27.9±1.7 b dis. Thus, Ca signaling mediated by CatSper1 and 2 may not a Sperm were incubated in medium with or without 2.4 mM added Ca for play a major role in initiation of motility in the presence of 90 min (mean±SEM). cAMP but rather in modulating the amplitude of the principal b Different from 2.4 mM Ca (P<0.05). flagellar bend. 220 B. Marquez et al. / Developmental Biology 303 (2007) 214–221

In conclusion, the results of this study suggest that, whereas Ho, H.C., Suarez, S.S., 2001. An inositol 1,4,5-trisphosphate receptor-gated 2+ internal Ca2+ stores could provide sufficient Ca2+ for the intracellular Ca store is involved in regulating sperm hyperactivated motility. Biol. Reprod. 65, 1606–1615. induction of hyperactivation, Ca2+ influx is required to maintain 2+ Ho, H.C., Suarez, S.S., 2003. Characterization of the intracellular calcium store intracellular Ca levels sufficient to sustain hyperactivation. at the base of the sperm flagellum that regulates hyperactivated motility. Also, the absence of CatSper1 and 2 may cause a deficiency of Biol. Reprod. 68, 1590–1596. Ca2+ influx necessary to maintain levels that promote normal Ho, H.C., Granish, K.A., Suarez, S.S., 2002. Hyperactivated motility of bull 2+ activated and hyperactivated flagellar beat patterns and there- sperm is triggered at the axoneme by Ca and not cAMP. Dev. Biol. 250, 208–217. fore, the inability of sperm to hyperactivate is not the primary Jungnickel, M.K., Marrero, H., Birnbaumer, L., Lemos, J.R., Florman, H.M., defect in CatSper1 and CatSper2 null mutants. 2001. Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3. Nat. Cell Biol. 3, 499–502. Acknowledgments Kirichok, Y., Navarro, B., Clapham, D.E., 2006. Whole-cell patch-clamp measurements of spermatozoa reveal an alkaline-activated Ca2+ channel. Nature 439, 737–740. We thank Drs. Timothy Quill and the late David Garbers, Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the

University of Texas Southwestern Medical Center, for pro- head of bacteriophage T4. Nature 227, 680–685. viding CATSPER heterozygote and homozygote null mutant Lindemann, C.B., Goltz, J.S., 1988. Calcium regulation of flagellar curvature male mice, sperm samples, and antibody to CatSper2. This and swimming pattern in Triton X-100-extracted rat sperm. Cell Motil. – work was supported by National Science Foundation grant Cytoskeleton 10, 420 431. Lindemann, C.B., Goltz, J.S., Kanous, K.S., 1987. Regulation of activation state 39034 to S.S.S. and NIH predoctoral fellowship to B.M. and flagellar wave form in epididymal rat sperm: evidence for the involvement of both Ca2+ and cAMP. Cell Motil. Cytoskeleton 8, 324–332. Appendix A. Supplementary data Marquez, B., Suarez, S.S., 2004. Different signaling pathways in bovine sperm regulate capacitation and hyperactivation. Biol. Reprod. 70, – Supplementary data associated with this article can be found, 1626 1633. Miyazaki, S., Shirakawa, H., Nakada, K., Honda, Y., Yuzaki, M., Nakade, S., in the online version, at doi:10.1016/j.ydbio.2006.11.007. Mikoshiba, K., 1992. Antibody to the inositol trisphosphate receptor blocks thimerosal enhanced Ca2+-induced Ca2+ release and Ca2+ oscillations in References hamster eggs. FEBS Lett. 309, 180–184. Mortimer, ST., Mortimer, D., 1990. Kinetics of human spermatozoa incubated Aoki, F., Sakai, S., Kohmoto, K., 1999. Regulation of flagellar bending by under capacitating conditions. J. Androl. 11, 195–203. cAMP and Ca2+ in hamster sperm. Mol. Reprod. Dev. 53, 77–83. O'Toole, C.M.B., Arnoult, C., Darszon, A., Steinhardt, R.A., Florman, H.M., Berridge, M.J., 1995. Capacitative calcium entry. Biochem. J. 312, 1–11. 2000. Ca2+ entry through store-operated channels in mouse sperm is initiated Berridge, M.J., Lipp, P., Boatman, M.D., 2000. The calcium entry pas de deux. by egg ZP3 and drives the acrosome reaction. Mol. Biol. Cell 11, Science 287, 1604–1605. 1571–1584. Braun, R.E., Behringer, R.R., Peschon, J.J., Brinster, R.L., Palmiter, R.D., 1989. Putney, J.W., 1986. A model for receptor-regulated calcium entry. Cell Calcium Genetically haploid spermatids are phenotypically diploid. Nature 337, 7, 1–12. 373–376. Quill, T.A., Ren, D., Clapham, D.E., Garbers, D.L., 2001. A voltage-gated Carlson, A.E., Westenbroek, R.E., Quill, T., Ren, D., Clapham, D.E., Hille, B., ion channel expressed specifically in spermatozoa. Proc. Natl. Acad. Garbers, D.L., Babcock, D.F., 2003. CatSper1 required for evoked Ca2+ Sci. U. S. A. 98, 12527–12531. entry and control of flagellar function in sperm. Proc. Natl. Acad. Sci. U. S. A. Quill, T.A., Sugden, S.A., Rossi, K.L., Doolittle, L.K., Hammer, R.E., Garbers, 100, 14864–14868. D.L., 2003. Hyperactivated sperm motility driven by CatSper2 is required Carlson, A.E., Quill, T.A., Westenbroek, R.E., Schuh, S.M., Hille, B., for fertilization. Proc. Natl. Acad. Sci. U. S. A. 100, 14869–14874. Babcock, D.F., 2005. Identical phenotypes of CatSper1 and CatSper2 null Ren, D., Navarro, B., Perez, G., Jackson, A.C., Hsu, S., Shi, Q., Tilly, J.L., sperm. J. Biol. Chem. 280, 32238–32244. Clapham, D.E., 2001. A sperm ion channel required for sperm motility and Castellano, L.E., Trevino, C.L., Rodriquez, D., Serrano, C.J., Pacheco, J., male fertility. Nature 413, 603–609. Tsutsumi, V., Felix, R., Darszon, A., 2002. Transient receptor potential Somanath, P.R., Jack, S.L., Vijayaraghavan, S., 2004. Changes in sperm (TRPC) channels in human sperm: expression, cellular localization glycogen synthase kinase-3 serine phosphorylation and activity accompany and involvement in the regulation of flagellar motility. FEBS Lett. 541, motility initiation and stimulation. J. Androl. 25, 605–617. 69–74. Stauss, C.R., Votta, T.J., Suarez, S.S., 1995. Sperm motility hyperactivation Clapham, D.E., 2003. TRP channels as cellular sensors. Nature 426, 517–524. facilitates penetration of the hamster zona pellucida. Biol. Reprod. 52, Esposito, G., Jaiswal, B.S., Xie, F., Krajnc-Franken, M.A.M., Robben, 1280–1285. T.J.A.A., Strik, A.M., Kuil, C., Philipsen, R.L.A., Van Duin, M., Suarez, S.S., Dai, X., 1995. Intracellular calcium reaches different levels of Conti, M., Gossen, J.A., 2004. Mice deficient for soluble adenylyl elevation in hyperactivated and acrosome-reacted hamster sperm. Mol. cyclase are infertile because of a severe sperm-motility defect. Proc. Reprod. Dev. 42, 325–333. Natl. Acad. Sci. U. S. A. 101, 2993–2998. Suarez, S.S., Osman, R.A., 1987. Initiation of hyperactivated flagellar bending Handrow, R.R., First, N.L., Parrish, J.J., 1989. Calcium requirement and in mouse sperm within the female reproductive tract. Biol. Reprod. 36, increased association with bovine sperm during capacitation by heparin. 1191–1198. J. Exp. Zool. 25, 174–182. Suarez, S.S., Katz, D.F., Overstreet, J.W., 1983. Movement characteristics and Herrick, S.B., Schweissinger, D.L., Kim, S.W., Bayan, K.R., Mann, S., acrosomal status of rabbit spermatozoa recovered at the site and time of Cardullo, R.A., 2005. The acrosomal vesicle of mouse sperm is a calcium fertilization. Biol. Reprod. 29, 1277–1287. store. J. Cell. Physiol. 202, 663–671. Tash, J.S., Means, A.R., 1982. Regulation of protein phosphorylation and Hess, K.C., Jones, B.H., Marquez, B., Chen, Y., Ord, T.S., Kamenetsky, M., motility of sperm by cyclic adenosine monophosphate and calcium. Biol. Miyamoto, C., Kopf, G.S., Suarez, S.S., Levin, L.R., Williams, C.J., Buck, Reprod. 26, 745–763. J., Moss, S.B., 2005. Bicarbonate responsive ‘soluble’ adenylyl cyclase is Towbin, H., Staehelin, T., Gordon, J., 1979. Electrophoretic transfer of proteins responsible for multiple specific signaling events in mammalian sperm from polyacrylamide gels to nitrocellulose sheets: procedure and some required for fertilization. Dev. Cell 9, 249–259. applications. Proc. Natl. Acad. Sci. U. S. A. 76, 4350–4354. B. Marquez et al. / Developmental Biology 303 (2007) 214–221 221

Trevino, C.L., Serrano, C.J., Beltran, C., Felix, R., Darszon, A., 2001. Wennemuth, G., Westenbroek, R.E., Xu, T., Hille, B., Babcock, D.F., 2000. 2+ Identification of mouse trp homologs and lipid rafts from spermatogenetic Cav2.2 and Cav2.3 (N- and R-type) Ca channels in depolarization-evoked cells and sperm. FEBS Lett. 509, 119–125. entry of Ca2+ into mouse sperm. J. Biol. Chem. 275, 21210–21217. Trevino, C.L., Felix, R., Castellano, L.E., Gutierrez, C., Rodriquez, D., Pacheco, Wiesner, B., Weiner, J., Middendorff, R., Hagen, V., Kaupp, U.B., Weyand, I., J., Lopez-Gonzalez, I., Gomora, J.C., Tsutsumi, V., Hernandez-Cruz, A., 1998. Cyclic nucleotide-gated channels on the flagellum control Ca2+ entry Fiordelisio, T., Scaling, A.L., Darszon, A., 2004. Expression and differential into sperm. J. Cell Biol. 142, 473–484. cell distribution of low-threshold Ca2+ channels in mammalian male germ Woolley, D.M., 2003. Motility of spermatozoa at surfaces. Reproduction 126, cells and sperm. FEBS Lett. 563, 87–92. 259–270. Ventela, S., Toppari, J., Parvinen, M., 2003. Intercellular organelle traffic Yanagimachi, R., 1994. Mammalian fertilization. In: Knobil, E., Neill, J. through cytoplasmic bridges in early spermatids of the rat: mechanisms of (Eds.), The Physiology of Reproduction. Raven Press, New York, haploid gene product sharing. Mol. Biol. Cell 14, 2768–2780. pp. 189–317. Walensky, L.D., Snyder, S.H., 1995. Inositol 1,4,5-trisphosphate receptors Yoshida, M., Ishikawa, M., Izumi, H., De Santis, R., Morisawa, M., 2003. Store- selectively localized to the acrosomes of mammalian sperm. J. Cell Biol. operated calcium channel regulates the chemotactic behaviour of ascidian 130, 857–869. sperm. Proc. Natl. Acad. Sci. U. S. A. 100, 149–154.